Electrophoretic separation of nucleic acid (DNA/RNA) strands based on fragment length (by means of a gel or capillary) is a widely used tool in laboratory genetics. This process is used to assess the distribution of fragment lengths in a DNA/RNA sample. Visualization of DNA/RNA after electrophoretic separation relies on the attachment of one or more dye molecules to the DNA/RNA. A dye molecule can be excited to fluorescence in order to ascertain the location of DNA/RNA within a gel column, channel or other laneway (hereinafter collectively “laneway” unless otherwise presented) along which the DNA/RNA has traversed via electrophoresis. Interpretation of the DNA/RNA fragment length distribution generally relies upon a comparison between at least one sizing reference, and preferably a plurality of sizing references, which consist of DNA/RNA of discrete, known sizes (“markers”). The implementation of markers can come in varying forms, but a common method to optimize sizing accuracy is to include the markers in the same laneway for electrophoresis as the fragmented DNA/RNA.
One currently preferred method for estimating the size of DNA/RNA fragments using electrophoresis comprises the inclusion of known markers within a sample of such fragments before commencing electrophoresis, i.e., use of internal markers. However, as both the DNA/RNA sample and markers usually incorporate the same dye to facilitate visualization thereof within the laneway, it can be difficult or impossible to distinguish between the two if there is a significant concentration of DNA/RNA fragments of the same size and with the same length(s) as the marker(s). In such situations, the fluorescence from the dye attached to the DNA/RNA sample fragments will often mask the fluorescence of the marker(s) dye. This consequence results inaccurate size estimation of the DNA/RNA fragment lengths insofar as the internal marker(s) cannot be distinguished to provide a reference for comparison.
The problem of masking the fluorescence signal of the internal marker can be largely avoided through selection of internal markers that are of a size that permits their discrimination from the sample. The Bioanalyzer 2100 capillary electrophoresis system (Agilent Technologies), for example, utilizes a two internal marker system with one being very small (˜15-50 bp) and the other being very large (˜1.5-17 kbp) to automatically assess the fragment length distribution of DNA/RNA. By selecting internal markers of divergent lengths, the chances of significant overlap of fragment lengths between the sample DNA/RNA and the markers are statistically minimized.
While the use of divergent length internal markers can circumvent the masking problem described above, the results of fragment length estimation are improved when the internal marker(s) are of a size that more closely approximates that of the sample: more accurate estimations of DNA/RNA fragment lengths can be provided by using more suitably sized internal markers, however, doing so increases the likelihood of undesirable fluorescence masking.
Another approach for estimating the size of DNA/RNA fragments using electrophoresis comprises the use of external markers, i.e., introducing markers into a laneway adjacent to the laneway(s) loaded with the DNA/RNA sample(s). After electrophoresis has been completed, estimations of fragment lengths in the sample laneway(s) can be made by comparing them to the external marker laneway.
External marker fragment sizes can be suitably chosen to match the fragment size ranges of the sample; there is no chance for any overlap or fluorescence saturation between them. However, the comparison is not optimal as variances in applied current and the laneway matrix composition between the external marker(s) and the DNA/RNA samples can confound sizing approximations. Additionally, the actual sample contents can impact fragment mobility, such that the migration speed of identical molecules can differ between channels depending upon the remaining sample composition. To account for variances and improve fragment length estimations, it is best to include the markers with the DNA/RNA sample in the same laneway.
Finally, another method for assessing fragment lengths involves labeling internal markers with fluorescent dyes with unique excitation/emission spectra on the ends of DNA/RNA molecules. This method has limitations though. Because dye molecules are only attached to the ends of the internal markers, any signal emitted by such a marker molecule is minimal, and can only be detected by highly sensitive, costly detectors that are not compatible with gel electrophoresis. Additionally, large markers (i.e., larger than 2,500 bp) have exceedingly high DNA/RNA fragment/dye molecule ratios. And, because the absolute mass of markers that can be loaded has a practical upper limit, this ratio prohibits the utilization of large internal markers even for analytical systems with highly sensitive detectors (i.e., capillary systems).